The ability to culture in vitro viable three-dimensional cellular constructs that mimic natural tissue has proven very challenging. One of the most difficult of the many problems faced by researchers is that there are multiple dynamic biochemical and mechanical interactions that take place between and among cells in vivo, many of which have yet to be fully understood, and yet the complicated in vivo system must be accurately modeled if successful development of engineered tissues in vitro is to be accomplished. The ideal in vitro system should accurately model the mechanical environment as well as the essential cellular interactions found during in vivo development while providing purity of the desired product construct so as to enable utilization of the product, for instance as transplantable tissue.
Many existing culture systems are simple well plate designs that are static in nature and do not allow for manipulation of the local environment beyond the gross chemical inputs to the system. As such, the development of more dynamic culture systems has become of interest, because it introduces the possibility of advantageously changing the local environment over the course of a cell culture experiment. However, known dynamic systems have not been widely implemented in the field of cell culture, as they are labor intensive, cost prohibitive, have configurations which limit their experimental flexibility and lack inter- and intra-lab comparability because there is no universal standard procedure.
In another aspect, there are many advantages to culturing cells in 3D (as opposed to historic 2D cell culture) that are being increasingly appreciated with a societal focus on higher and higher fidelity in vitro models of in vivo human physiology. One of these many advantages relates to the cultured cells' phenotype. It is known that conventional 2D culture of cells is often associated with a loss of phenotype and cell damage while 3D culture has been shown to retain cell phenotype. See Mayne, R. et al (1976) PNAS, 73, 5; Brodkin, K. R. et al (2004) Biomaterials, 25, 28; Elowsson, L. (2009) PhD Thesis (University of Sheffield, UK); Benya, P. D. and Schaffer, J. D. (1982) Cell, 30, 1; Bonaventure, J. et al (1994) Experimental Cell Research, 212, 1; and Osiecka, I. et at (2008) Molecular Medicine Reports, 1, 6. However, current technology does not allow for the exploitation of 3D culture advantages requires innovation to address the practical difficulties of 3D culture when compared to its simpler, 2D cell culture, predecessor.
One such exemplary technology lag area has been the process of cell passaging, in which a relatively small number of cells are repeatedly doubled for the sole purpose of creating a large number of cells (e.g., to achieve the number of cells necessary for a particular experiment). As one skilled in the art will appreciate, passaging cells in 2D is convenient and ubiquitously standardized. Additionally, during conventional passaging cells in 2D procedures, most cells enter into a state of rapid proliferation which decreases the time necessary to achieve the desired large number of cells. As one skilled in the art will appreciate, in conventional 2D passaging, cells respond to the stiffness of the material on which they attach. See, Attachment A, Micro- and Nanoengineering of the Cell Microenvironment, Technologies and Applications (Engineering in Medicine & Biology), All Khademhosseini (Editor)).
Relative to tissues found in the body formed from organic materials, tissue culture lab ware is typically formed from stiff materials. For example and without limitation, the moduli of soft mammalian tissues ranges from about 100 Pa to about 950 kPa. Exemplary moduli of soft mammalian tissues include:
TABLE 1A summary of elastic moduli of several different tissues. Experimentalelastic moduli of a variety of tissues. including the animal oforigin of the tissue, and the testing modality used to determinethe modulus. When multiple stiffness values were available. thevalue at the lowest strain rate and lowest pre-strain was usedto approximate the “resting stiffness” of the tissueTestingElasticTissue typeAnimalmethodmodulusRefAchilles' tendonRatTension310Mpa15Articular cartilageBovineCompression950kPa86Skeletal muscleRatTension100kPa87Carotid arteryMousePerfusion90kPa88Spinal cordHumanTension89kPa89Thyroid canceraHumanCompression45kPa16Spinal cordRatTension27kPa90Cardiac muscleMouseTension20-150kPa91Skeletal muscleMouseAFM12kPa13ThyroidHumanCompression9kPa16LungGuinea pigTension5-6kPa5Breast tumorHumanCompression4kPa7KidneySwineRheology2.5kPa92Premalignant breastbHumanIndentation2.2kPa14Fibrotic liverHumanCompression1.6kPa93LiverHumanCompression640Pa93Lymph containingHumanVibrational330Pa17metastasesresonanceBrainSwineIndentation260-490Pa94Lymph NnodeHumanVibrational120Pa17resonanceMammary glandHumanCompression160Pa7FatHumanIndentation17Pa14aThyroid papillary adenocarcinoma.bMammary ductal carcinoma in situ.2 | Soft Matter, 2006, 2, 1-9This journal is  © The Royal Society of Chemistry 2006See, Soft Matter, 2007, 3, 299-306, DOI: 10.1039/b610522j“Soft biological materials and their impact on cell function” Ilya Levental, Penelope C. Georges and Paul A. Janmey.
Exemplary tissue culture lab ware formed from polystyrene has an elastic modulus of 3-3.5 GPa, which is higher than the modulus of tissues formed from organic materials but not as high as the elastic modulus of bone (9 GPa). In this aspect, bone is a composite made up of inorganic minerals with high bulk moduli and organic materials which are much softer, and the contribution of the inorganic materials increases the modulus correspondingly. See, Journal of Biomedical Materials Research Part A Volume 67A, Issue 3, Pages 886-899 Published Online: 20 Oct. 2003, (bulk hydroxyl apatite modulus of 34-117 GPa).
Currently there are no commercially available products designed for 3D cell passaging. Published research on this topic to date has explored aspects of the potential use of 3D hydrogels (of hyaluronic acid and poly(NIPAM) respectively) for 3D cell passaging and has shown benefits of phenotype retention. See, TERMIS-EU 2010 Oral Presentation “Thermally-responsive Polymers for 3D Chondrocyte Culture;” and U.S. patent application Ser. No. 11/473,870 to Singh, which is incorporated herein by reference in its entirety. However, hydrogel matrices are not in the stiffness range of tissue culture polystyrene or bone.
What is needed in the art is a method for culturing cells in a dynamic environment in which the physical and biochemical conditions can be advantageously changed over the course of time. Moreover, what is needed is a system in which cells can be developed to form a three-dimensional construct, while maintaining the isolation and purity of the developing product cells. In another aspect, what is needed is a material and method for 3D cell passaging that the use of a stiff culture material in a 3D cell culture environment while maintaining a desired level of phenotype retention.